Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts

Bacterial Manipulations of the Host Cell Proteome Michael Starnbach . Harvard Medical School, Boston, USA.

Intracellular bacterial pathogens directly alter host cells in order to survive. While microarray analysis has been used to identify changes to host transcription during infection, it has remained difficult to catalog post-transcriptional alterations that also occur. We have applied the global protein stability (GPS) platform and quantitative proteomics to identify changes in host protein stability following infection with the obligate intracellular pathogen Chlamydia trachomatis. Our results suggest that C. trachomatis profoundly remodels the host proteome independently of changes in transcription and some of these regulated proteins are essential for bacterial replication. These direct proteomic approaches can now be applied to examine a broad range of host-pathogen interactions and to begin developing potential host-based therapeutics. On the Mechanism of the Amidases – Consequences for Sulfhydryl Catalysis and Drug Design B T. Sewell , S W. Kimani, R Hunter. University of Cape Town, Cape Town, South Africa. The amidases, are thiol enzymes that catalyze the hydrolysis and condensation of non-peptidic amide functional groups. Amidases occur in both prokaryotic and eukaryotic organisms, where they play important physiological roles including the synthesis of metabolites, protein post- translational modification, vitamin and co-enzyme metabolism and protein and amino acid deamination. Enzymes in this family find application as biocatalysts in the fine chemical industry, and as tools for drug synthesis. In addition, the glutamine dependent NAD+ synthetase in Mycobacterium tuberculosis, which contains an amidase domain, has been suggested as a potential drug target. We have studied the mechanism of a model amidase from a psychrophile: Nesterenkonia species (NitN). The enzyme has four catalytically essential residues: a cysteine, two glutamates and a lysine. We have investigated the geometry and interactions in the active site using X-ray crystallography and quantum mechanical modeling. Our observations strongly indicate that the generally accepted view that assistance by a glutamate acting as a general base catalyst is needed for thiolate generation in order for the nucleophilic addition step to proceed, is incorrect. Our results clearly demonstrate that realization of stereoelectronic alignment of the sulphur lone pair orbital and the amide LUMO allows the initial nucleophilic addition step of the hydrolysis to proceed via a neutral thiol (sulfhydryl group), so long as there is general acid catalysis on the carbonyl oxygen. In this regard the reaction, rather than being driven primarily by the nucleophilicity of the sulfur as a thiolate, may proceed via a combination of neutral sulfur nucleophilicity with stereoelectronic alignment in tandem with mild carbonyl electrophilicity enhancement as a result of the general acid catalysis.

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